Phosphodiesterase type 5 inhibitors enhance chemotherapy in preclinical models of esophageal adenocarcinoma by targeting cancer-associated fibroblasts

Summary The chemotherapy resistance of esophageal adenocarcinomas (EACs) is underpinned by cancer cell extrinsic mechanisms of the tumor microenvironment (TME). We demonstrate that, by targeting the tumor-promoting functions of the predominant TME cell type, cancer-associated fibroblasts (CAFs) with phosphodiesterase type 5 inhibitors (PDE5i), we can enhance the efficacy of standard-of-care chemotherapy. In ex vivo conditions, PDE5i prevent the transdifferentiation of normal fibroblasts to CAF and abolish the tumor-promoting function of established EAC CAFs. Using shotgun proteomics and single-cell RNA-seq, we reveal PDE5i-specific regulation of pathways related to fibroblast activation and tumor promotion. Finally, we confirm the efficacy of PDE5i in combination with chemotherapy in close-to-patient and in vivo PDX-based model systems. These findings demonstrate that CAFs drive chemotherapy resistance in EACs and can be targeted by repurposing PDE5i, a safe and well-tolerated class of drug administered to millions of patients world-wide to treat erectile dysfunction.


In brief
Resistance to standard-of-care chemotherapy in esophageal adenocarcinoma is dependent on cancer cell extrinsic mechanisms of the tumor microenvironment. Sharpe et al. show that repurposing PDE5 inhibitors to target the tumor-promoting function of cancerassociated fibroblasts enhances the efficacy of chemotherapy in 3D-tumor models INTRODUCTION Esophageal adenocarcinoma (EAC) is usually lethal. Most patients present with late-stage disease, and for those amenable to potentially curative treatments, 5-year survival is 50% at best. Randomized controlled trials (RCTs) confirm a survival advantage for neoadjuvant chemotherapy with or without radiotherapy, but this benefit is restricted to a minority of patients. For the majority, neoadjuvant treatments are ineffective, are morbid, and delay definitive surgery. [1][2][3] Large-scale genome-sequencing studies have detailed the genetic landscape of EAC and identified potential molecular targets. These data reveal a highly complex tumor with driver gene mutations present in non-malignant precursor lesions that never progress to cancer, suggesting that drivers of disease development and progression may lie within the tumor microenvironment. [4][5][6][7][8] We have previously reported that activated cancer-associated fibroblasts (CAFs) influence outcome in EAC and the biological properties of esophageal CAFs that promote tumor progression. 9, 10 CAFs have also been shown to influence the immune cell infiltrate and response to chemotherapy in a range of tumors. [11][12][13] In general, the tumor-promoting properties of CAFs have been associated with the alpha-smooth muscle actin (a-SMA)-positive, activated myofibroblast phenotype observed in cancer, fibrosis, and wound healing. 14-16 CAF-targeting strategies have mostly focused on the effectors of CAF tumor promotion including cell signaling and extracellular matrix (ECM) molecules. We have been working to understand how to target the CAF phenotype itself and whether new or existing drugs can be purposed for this use.
Phosphodiesterase type 5 (PDE5) is part of a complex superfamily of hydrolases that control cAMP and cGMP levels by catalyzing their breakdown. 17 PDE5 is widely expressed in normal tissue and many human cancers, and its inhibition results in an upregulation of cGMP, which activates several downstream pathways including protein kinase G (PKG) signaling. Downstream substrates of PKG are implicated in a variety of biological processes such as smooth muscle contraction, cell differentiation, proliferation, adhesion, and apoptosis. 18,19 The main function of PDE5 is to control vascular tone by regulating intracellular cGMP and calcium levels.
Phosphodiesterase type 5 inhibitors (PDE5i) were first licensed to treat erectile dysfunction. More recently, high doses have been approved to treat pulmonary arterial hypertension and lower urinary tract symptoms. 20-23 New studies suggest repurposing PDE5i for treating conditions such as cancer or lung disease. 19,24 PDE5i have been found to attenuate the myofibroblast phenotype of prostatic fibroblasts, suggesting that they could target the inflammatory/activated microenvironment observed in many solid tumors. 25 We hypothesized that, in EAC, directly targeting the CAF phenotype with PDE5i would downregulate the tumor-promoting effects of CAFs and improve EAC sensitivity to conventional chemotherapy. This may improve outcomes for patients with EAC, of whom up to 80% do not respond to standard-of-care neoadjuvant treatment. 26 Recent evidence has shown that multimodal therapies of this type have acceptable tolerability and therapeutic potential. [27][28][29] In this study, we characterized PDE5 expression in the human esophagus and described the effect of PDE5i on the tumor-promoting functions of esophageal CAFs in 2D and 3D models in vitro. We documented changes in CAF protein expression in response to PDE5i using shotgun proteomics and applied single-cell RNA sequencing to demonstrate a phenotypic change in CAFs driven by co-culture with cancer cells and inhibited by PDE5i treatment. Finally, we moved to a validated near-patient EAC model system to assess tolerability and efficacy of PDE5i in combination with standard-of-care chemotherapy and tested the safety and efficacy of this combination in a murine model.

Characterization of PDE5i in esophageal cancer
To assess the suitability of PDE5 as a target in EAC, we determined the expression of PDE5A in EAC relative to normal tissue, esophageal squamous cell carcinoma (ESCC), and the EACrelated pre-cursor condition Barrett's esophagus (BE) in publicly available gene expression datasets with matching tissue samples. PDE5A was differentially expressed between ESCC (n = 9), EAC (n = 21), and BE (n = 20) samples compared with normal esophageal squamous epithelium (n = 19) 30 (one-way ANOVA, p < 0.0001; Figure 1A). Specifically, PDE5A was significantly overexpressed in EAC, BE, and ESCC versus normal squamous epithelium (p < 0.0001). This upregulation of PDE5A expression in EAC and BE compared with normal adjacent esophageal epithelium was confirmed in another publicly available dataset (n = 48, 5, and 18 respectively) 31 ( Figure 1B, one-way ANOVA, p < 0.0001) and by comparing RNA-seq data from EAC samples in TCGA (n = 85) 32 with normal esophageal squamous epithelium in the GTEx database (n = 269) 33 ( Figure 1C; Welch's t test, p < 0.0001). Using publicly available data in R2 for EAC, 34 we found that PDE5A expression was associated with worse overall survival ( p = 0.023; Figure 1D).
Next, we assessed PDE5 protein expression in EAC resection specimens, normal esophageal tissue, EAC tumor cell lines, and CAFs derived from primary resected tumor tissue. In keeping with the gene expression data, PDE5 was highly and ubiquitously expressed in esophageal cancer cells and surrounding stroma compared with low expression in normal esophageal squamous epithelium ( Figure 2A). In matched normal esophageal fibroblasts (NOFs) and CAFs, two commonly used EAC cell lines and a primary epithelial esophageal cancer cell line extracted in our laboratory (MFD-1), 36 variable PDE5 expression was observed, as determined by cell sub-type. We observed the highest PDE5 expression in CAFs, with little or no PDE5 protein expression in EAC cell lines ( Figure 2B; all normalized to Hsc70). Although in general a heterogeneous population, CAFs are associated with a contractile and secretory phenotype characterized by increased expression of a-SMA. 37 This activated, myofibroblastic state in cancer is believed to be driven by cancer cell signaling and can be recapitulated by treating normal fibroblasts with TGF-b1 in vitro. 38 Importantly, when one is considering any future clinical application of PDE5i in cancer treatment, it would be vital to demonstrate that not only can PDE5i revert the established CAF phenotype but also PDE5i treatment can prevent the transdifferentiation of resident NOFs to CAF. Therefore, we repeated our previous experiments to confirm that NOFs treated with TGF-b1 significantly induced a-SMA expression, 9 but when cotreated with 50 mM vardenafil (a specific PDE5i) the increase in a-SMA expression was abolished ( Figure 2C). This dose of PDE5i is high compared with its reported IC 50 of 0.7 nM 39 but is consistent with other studies reporting inhibition of PDE5 to target myofibroblast differentiation in fibroblast cultures with micromolar-scale concentrations of vardenafil, 25,40,41 likely reflecting the strong myofibroblastic phenotype observed when grown in ex vivo culture conditions. Having established the potential of PDE5i to prevent NOF transdifferentiation in vitro, we explored the possibility that PDE5i could suppress a-SMA expression in CAFs. After 72 h of culture with vardenafil, CAFs reduced a-SMA expression by over 50% (p < 0.01; Figure 2D). The ontarget effects of PDE5i were confirmed by observing appropriate decreases in PDE5 and a-SMA protein expression in response to PDE5 siRNA (Figures S1A-S1C). Importantly for potential in vivo applications, we found that daily dosing of PDE5i produced significant a-SMA downregulation compared with a single application of PDE5i-containing medium 72 h before analysis (Figure S1D). After withdrawal of PDE5i, a-SMA expression returned to pre-treatment levels within 72 h ( Figure S1E).

PDE5 inhibition reduces fibroblast contraction and esophageal cancer cell invasion in vitro
The expression of a-SMA is characteristic of the myofibroblast phenotype but does not necessarily indicate functional capacity.
To test the hypothesis that PDE5i treatment of myofibroblasts could interfere with known tumor-promoting functions, we performed a series of in vitro experiments to assess ECM contraction and the promotion of cancer cell invasion. NOFs were embedded in collagen-1 gels after being treated with TGF-b1 alone or with TGF-b1 + vardenafil for 72 h. Fibroblasts that were treated with TGF-b1 significantly increased both a-SMA expression and collagen-1 gel contraction, but after pretreatment with vardenafil the induction of a-SMA and gel contraction was substantially and consistently reduced ( Figure 3A). CAFs express high levels of a-SMA and induce collagen-1 gel contraction. Treatment with vardenafil significantly reduced a-SMA expression and collagen-1 gel contraction in CAFs ( Figure 3B). Next, we assessed the ability of conditioned medium taken from fibroblast cultures to promote cancer cell invasion in transwell invasion assays under a variety of conditions, as previously described. 9 The conditioned medium from TGF-b1-treated NOFs promoted five times more invasion of EAC cells than conditioned medium from vehicle-treated NOFs, whereas when vardenafil was added to TGF-b1 treatment of NOFs, the resulting conditioned medium did not promote invasion ( Figure 3C). Similarly, vardenafil-treated CAF-conditioned medium induced significantly less cancer cell invasion than vehicle-treated CAFconditioned medium ( Figure 3D). Similar observations were made using PDE5 siRNA ( Figure S1). This finding was reproduced in the more physiologically relevant organotypic co-culture model, where we observed that vardenafil-treated CAFs had lost their ability to promote cancer cell invasion compared with vehicle-treated CAF ( Figure 3E). These findings suggested that, in vitro, PDE5i treatment was able to suppress both the transdifferentiation of NOFs and the tumor-promoting characteristics of CAF that we had previously observed. 9 Proteomic analysis of fibroblasts treated with vardenafil or PDE5 siRNA identifies modulation of major pathways associated with cancer promotion In keeping with previous reports on benign disease, 25 we established the ability of PDE5i to ameliorate some of the tumorpromoting functions of TGF-b1-driven, activated esophageal fibroblasts in vitro. To explore the cellular events responsible for these effects, we took a whole-proteome-based approach. We have previously demonstrated the benefits of this approach to identify pathways and participating proteins that may provide novel insight into the tumor-promoting properties of CAFs. 42 Proteomic analysis was carried out on a representative NOF/ CAF patient-matched pair. The CAFs were treated with vehicle (negative control), vardenafil (PDE5i), PDE5 siRNA (positive control), and negative control siRNA and total protein expression assessed by quantitative proteomic profiling. To examine the effects of vardenafil or PDE5i siRNA treatment on the global proteomic profile of CAFs, we considered the following log2 ratios: PDE5i versus CAF vehicle, PDE5 siRNA versus siRNA negative control, and CAF vehicle versus NOF. In total, 8,118 proteins were quantified across all analyzed samples (peptide level FDR < 0.05; Table S1). Principal component analysis of all quantified proteins showed that vardenafil-treated CAFs clustered together with PDE5 siRNA-treated CAFs compared with vehicle-treated CAFs ( Figure 4A). Since their global proteomic  profiles were similar, we considered PDE5i versus CAF vehicle and PDE5 siRNA versus siRNA negative control as one group and performed a one-sample t test to identify differentially expressed proteins (DEPs) following treatment with vardenafil or PDE5 siRNA. In total, 812 proteins were found to be up-regulated and 725 down-regulated in CAFs treated with vardenafil or PDE5 siRNA compared with their respective controls (Table S2). In order to identify which of these proteins reflected the amelioration of the CAF phenotype following vardenafil or PDE5 siRNA treatment, we compared the DEPs in CAFs treated with vardenafil or PDE5 siRNA to our previously published dataset of DEPs in CAFs versus NOFs. 42 Using this approach, we identified 83 proteins that were down-regulated in CAFs versus NOFs but became up-regulated in CAFs following treatment with vardenafil or PDE5 siRNA ( Figure 4B). Conversely, we identified 88 proteins that were up-regulated in CAFs versus NOFs but became down-regulated in CAFs following treatment with vardenafil or PDE5 siRNA ( Figure 4B) (Table S3). We then performed gene ontology analysis for those 171 proteins that reversed their trend of modulation following treatment with vardenafil or PDE5 siRNA compared with CAFs. ECM organization (p = 0.01), ECM disassembly (p = 0.0008), sequestering of TGF-b in ECM (p = 0.0003), regulation of extracellular exosome assembly (p = 0.0005), cell-cell adhesion (p = 0.01), cell migration (p = 0.02), DNA damage response (p = 0.003), regulation of apoptosis (p < 0.0001), programmed cell death (p = 0.002), angiogenesis (p = 0.02), response to hypoxia (p = 0.02), and insulin receptor signaling pathway (p = 0.006) were significantly over-represented gene ontology (GO) terms ( Figure 4C). These are all major pathways associated with the cancer-promoting properties of fibroblasts and identified in our previous studies of EAC fibroblasts. 42 To provide additional granularity, proteins exhibiting the most significant changes in expression in response to PDE5i treatment have been represented on a heatmap with the corresponding GO term highlighted ( Figure 4D).
In summary, these findings suggest that vardenafil is specific for PDE5 inhibition in ex-vivo esophageal CAFs and leads to down-regulation of established cancer-promoting CAF pathways.
Single-cell RNA sequencing reveals suppression of activated CAF phenotypes in MFD-1/CAF co-cultures treated with a PDE5 inhibitor To this point, experiments had focused on understanding the specificity of PDE5i in prevention of fibroblast transdifferentiation and the functional/phenotypic effects of PDE5i on CAFs. To be useful as a potential CAF-targeting treatment in cancer, these effects would need to be retained in the presence of cancer cells and be able to overcome any cancer cell-derived CAF-promoting signaling. To explore this, we took a single-cell whole-transcriptomic approach using droplet-based microfluidics and single-cell RNA sequencing (DropSeq) to analyze the gene expression of individual CAFs and esophageal cancer cells in direct co-culture. 43 CAFs were grown in isolation or in co-culture with the esophageal cancer cell line, MFD-1. 36 Cells were treated with vardenafil for 72 h before analysis, where indicated. This model was used to look at the transcriptional regulation of both cancer cells and CAFs in the presence of PDE5 inhibition.
Unsupervised clustering produced two broad clusters of cells, identified as either MFD-1 (cancer cells) or CAFs, as defined by their transcriptomic profiles ( Figure 5A). Within these clusters a further eight sub-clusters were identified ( Figure 5B). In general, the cells clustered on the basis of their cell type and within those clusters on their culture conditions.
To characterize the individual sub-clusters of cell phenotypes, differential gene expression analysis was performed using Seurat's FindAllMarkers function with a log fold change cut-off of 1 and otherwise default settings. Canonical marker genes differentially expressed between populations of CAFs (Thy-1 Figure 5C. The most striking finding from this experiment was the differential expression pattern of CAFs grown in different culture conditions. All CAFs from monoculture were similar in their transcriptomic profiles to each other ( Figure 5B; CAFp and CAFv). Importantly, the PDE5i-treated CAFs from monoculture (CAFp) and co-culture (CAF.CoC.p) clustered together, whereas the CAFs co-cultured with MFD-1 in the absence of PDE5i formed a distinct cluster ( Figure 5B, CAF.CoC.v). This cluster was markedly different to those from the monoculture with vehicle treatment and all other CAFs, indicating a phenotypic change in CAFs driven by co-culture with cancer cells and inhibited by PDE5i treatment.
The gene expression that defined the transcriptome of CAFs in co-culture is highlighted in Figure 5D. All are associated with the activated/myofibroblast phenotype of CAFs. These data demonstrate that CAFs adopt a myofibroblastic phenotype in coculture with cancer cells in vitro and that this process can be inhibited by treatment with PDE5i, despite the presence of cancer cells, resulting in down-regulation of myofibroblast genes such as ACTA2 (a-SMA), myosin light-chain kappa (MYLK), osteonectin (SPARC), and transgelin (TAGLN) ( Figure 5D).
3D co-culture models of close-to-patient cancer cells and human mesenchymal stem cells reveal that PDE5 inhibition increases the efficacy of chemotherapy in EAC Having established that the myofibroblast phenotype can be suppressed in 2D culture by using high concentrations of PDE5i, we sought to confirm our findings in more representative pre-clinical models. Pre-treatment biopsy tissue can be used to grow a patient's own cancer epithelial cells ex vivo in the recently established 3D tumor growth assay (3D-TGA), providing a platform for near-patient drug sensitivity assessment. 44 We used this platform to test the effect of PDE5i on EAC tumor cell sensitivity to platinum-based triplet (ECF) chemotherapy using 15 samples from eight different patients, with drug combinations at human tissue-relevant concentrations. Human mesenchymal stem cells (hMSCs) were included in the 3D-TGA, and these take up a myoCAF phenotype in culture and may be a source of CAFs in cancer. [45][46][47] In keeping with our previously published data, 44 only when cancer cells were grown with stromal support (hMSC co-cultures) did the models accurately predict the tumor regression grade (TRG) 48 observed in the patients from whom the cancer cells had been taken ( Figure 6A, lack of response to ECF represented by red). PDE5i treatment did not have toxic effects on hMSCs or EAC tumor cells, either alone or together in the 3D-TGA ( Figure S2). As previously observed, chemotherapy IC 50 was increased in co-culture compared with the no-hMSC controls (monoculture). There was no change in IC 50 with the addition of PDE5i to ECF in monoculture, but the addition of PDE5i in co-culture resulted in a significant reduction in the IC 50 (p = 0.0033; Figures 6A and 6B), to a level similar to or less than the mean peak serum concentration used in clinical practice. A PDE5i-mediated reduction in chemoresistance was found to be patient specific, with the size of the PDE5i chemo-sensitizing effect varying between patients ( Figure 6A); there was a trend toward a reduction in IC 50 seen in 12 of the 15 samples and a statistically significant (CI > 95%, p < 0.05) reduction in the IC 50 chemo-resistance in six of these samples. This suggests that ECF-resistant tumors could become sensitive to standard-of-care chemotherapy with the adjunctive administration of PDE5i. In order to understand the apparent lack of response to adjunctive PDE5i in some of the 3D-TGA models, we performed bulk RNA-seq on three patients' tumor samples and corresponding 3D-TGAs with and without hMSCs (OES4R, 5R, and 7R). The RNA-seq data from the 3D-TGAs was analyzed for the presence of canonical myo-CAF genes to enable a direct comparison with the native tumor of origin. In OES4R and 7R, we saw up-regulation of myoCAF genes in co-culture ( Figure 6C). In both, ECF resistance was induced in co-culture, and resistance was reduced with PDE5i. In the non-responder (5R), we did not detect myoCAF genes up-regulated in co-culture with hMSCs ( Figure 6C), and the 3D-TGA with PDE5i remained resistant to ECF ( Figure 6A). This suggests either that the hMSCs did not adopt a myoCAF phenotype or that they were absent and that 5R was resistant to ECF independently of stromal support.
In 9 of 12 3D-TGA cultures from the five patients whose tumors demonstrated a poor response to chemotherapy in the clinic (TRG 4/5), the addition of PDE5i resulted in complete or partial response to ECF at doses equivalent to those observed in  Figure 6A), and all five patients had at least one 3D-TGA with a response. Our near-patient experiments suggest that up to 75% (9/12) of resistant tumors could be rendered sensitive to standard chemotherapy by targeting myoCAFs with PDE5i.
PDE5i is safe and effective in combination with standard-of-care chemotherapy in esophageal patientderived xenograft (PDX)-bearing mice Before considering a human trial of PDE5i in EACs, we performed a dose-escalation study to assess potential serious toxicity of combining chemotherapy with PDE5i and an efficacy study in a PDX mouse model supplemented with human stromal support. The PDX was developed with esophageal cancer tissue taken from patient tumor sample Oes7R. This was chosen as it was resistant to chemotherapy clinically and in the 3D-TGA but was responsive to adjunctive PDE5i. Since human stroma is lost in PDX models over time, hMSCs were incorporated at passage to maintain a human stroma. A dose-finding study was initially performed with epirubicin, cisplatin and capecitabine (the oral equivalent of 5-FU) (ECX) in non-tumor-bearing mice (n = 3), using maximum doses equivalent to those used in humans (Table S4). This revealed peak tolerable doses of ECX that were 50% of the human equivalent doses ( Figure S3). Next, dose escalation studies for PDE5i were carried out in four groups of PDX-bearing mice (no treatment [n = 2], ECX alone [n = 2], ECX + PDE5i [vardenafil, n = 3], and ECX + PDE5i Figure 6. Modeling of patient response to chemotherapy with PDE5i using 3D-TGA. Sensitivity of close-to-patient cells was determined in 3D-TGA, with and without mesenchymal cell co-culture, after 4-day exposure to ECF and vardenafil (PDE5i) drug combinations (A) Viability curves were generated and IC 50 values determined for a cohort of EAC patients' ECF-treated 3D-TGAs, with (+) and without (À) hMSC support and the addition of PDE5i (n = 15 patient samples from 8 patients). The patient cancer cell clusters were classified as sensitive (green), borderline (orange), or resistant (red) by comparison of IC 50 values with the mean peak serum concentrations achieved in patients at the doses used in UK clinical practice. This is marked with an asterisk, where the IC 50 drop is significant (CI > 95%, p < 0.05). Tumor regression score (TRG) denotes the chemotherapy response of the patient's tumor clinically (TRG1-3, sensitive; 4-5, non-responsive). (B) Overall sensitivity of all the EAC patient samples co-cultured with hMSCs was determined for assays with and without the addition of PDE5i to ECF chemotherapy. Horizontal lines represent mean IC 50 s. [tadalafil, n = 3]). PDE5i dose increases were carried out in three phases in combination with a static dose of ECX (as previously determined). No adverse side effects (e.g., weight loss or lowered threshold of ECX tolerability) were reported for maximum doses of either vardenafil or tadalafil. Representative sections of native spleen, liver, aorta, and heart were assessed and showed no gross morphological differences between groups ( Figure S4), suggesting no deleterious effects with the addition of PDE5i. However, some slight weight loss in the ECX group was attributed to the use of epirubicin. Given this concern, and in line with animal welfare best practice, we conducted an additional study with cisplatin and capecitabine only (CX) at 75% of the doses used in the previous study, which was well tolerated.
Having identified the most appropriate standard-of-care regimen for our PDX models, we carried out an efficacy study with a larger cohort of mice in four groups as before (n = 15/group), with one group receiving saline for injection and the others receiving a static dose of CX with or without three cycles of PDE5i vardenafil or tadalafil (see STAR Methods). A significant reduction in tumor volume was observed in the CX, CX + Vardenafil, and CX + Tadalafil groups ( Figure 7A, two-way ANOVA, p values of < 0.003, < 0.0001, and < 0.007, respectively). Comparison of the tumor volume in the 4 groups on the final day of the study revealed that only the CX + Vardenafil group was significantly reduced compared with vehicle control (Kruskal-Wallis test, p = 0.04), demonstrating the stronger effect of CX + Vardenafil compared with ECX treatment alone or CX + Tadalafil. We also assessed effects of PDE5i treatment on CAF differentiation in vivo by conducting immunohistochemistry (IHC) for the CAF markers a-SMA and periostin in PDX tumor sections. We quantified changes in CAF markers by scanning these slides and digitally assessing the proportion of tissue stained by IHC relative to total tissue area in these treatment groups. CX treatment alone had no effect on a-SMA or periostin expression in PDX tumors compared with untreated mice (p = 0.31 and 0.87, respectively), but both PDE5i (vardenafil and tadalafil) were observed to  Figures 7B and 7C). CX + Vardenafil significantly reduced the proportion of periostin-positive stroma, whereas a-SMA was affected to a lesser extent ( Figures 7D  and 7E, p = 0.035 and 0.095, respectively). CX + Tadalafil significantly reduced the proportion of a-SMA-positive stroma ( Figures 7B and 7D, p = 0.01) but not periostin-positive stroma ( Figures 7C and 7E, p = 0.38).

DISCUSSION
Stromal remodeling can promote cancer progression. CAFs display an activated myofibroblast phenotype and expression of a-SMA in many solid tumors is a marker of reduced disease free and overall survival. 9,38,49-51 The tumor-promoting biology of CAFs make them a target for novel cancer therapies. In this study, we have demonstrated that PDE5 is a potential target for altering the fibroblast phenotype in EACs. Using a combination of conventional in vitro molecular biology techniques, and state-of-the-art proteomic and single-cell sequencing technologies, we have documented the specificity of PDE5i for fibroblasts both to prevent transdifferentiation of normal fibroblasts and to reverse the activated myofibroblast (CAF) phenotype. Finally, in a step toward a clinical trial, we have confirmed the efficacy of PDE5i in combination with chemotherapy in close-to-patient in vitro and in vivo PDX-based model systems.
Our findings are in keeping with many reports documenting the role of fibroblasts in cancer. Activated myofibroblasts are contractile and pro-invasive in EAC models. 9 There is evidence that CAFs can protect cancer cells from chemotherapy, 52 create an immunosuppressive environment, reduce the immune infiltrate, and alter the immune composition, allowing cancer cells to escape immune surveillance. 53-55 By reducing the transdifferentiation of fibroblasts in cancer and by modulating the phenotype of activated CAFs, we may be able to improve overall survival by several different mechanisms: improving response to chemo/immunotherapy, increasing tumor cell recognition by the immune system, and reducing cancer cell invasion.
Several strategies have been proposed to target pro-tumorigenic CAF functions, mostly through modulating the effectors of the CAF phenotype rather than the cell state itself. These include targeting the ECM-remodeling enzymes such as the lysyl oxidase family and MMPs, or targeting CAF-derived molecular signals (e.g., CXCR4, TGF-b, HGF; reviewed in Orsulic et al. 10 ).
Initial attempts to specifically target CAFs have centered on the membrane bound glycoprotein fibroblast activation protein alpha (FAP). Early promise with FAP-targeting monoclonal antibodies has not translated into clinical success (reviewed in Lindner et al. 56 ). Novel mechanisms to prevent myofibroblast differentiation and CAF accumulation are now required. PDE5i might offer a compelling way forward for this purpose. Importantly, PDE5i are a safe and well-tolerated class of drug administered to millions of patients world-wide to treat erectile dysfunction, benign lower urinary tract symptoms, and pulmonary arterial hypertension. 22,57 High-dose PDE5i show safety and efficacy for treating heart failure with reduced ejection fraction. 58 There is a significant body of evidence supporting PDE5i use in treating a range of cancers (reviewed in Pantziarka et al. 19 ). In particular, animal studies suggest that PDE5i have potent immunomodulatory activity that warrants clinical study with or without immune check-point inhibition. 59 PDE5i are currently being tested in combination with standard-of-care and other novel treatments in a range of cancer types, including gliomas, head and neck squamous cell cancer, pancreatic cancer, and malignant melanoma. 19 With this background, the sensible next steps for testing PDE5i in EAC are in the context of a phase I/II human clinical trial.
In summary, we provide in vitro, near-patient, and in vivo evidence for the potential role of PDE5i in treating esophageal adenocarcinoma and suggest a rationale for future human trials.

Limitations of the study
This study is not without shortcomings. Some of the in vitro work has been performed with cell lines that may not represent the true in vivo biology of these cell types. We supplemented these findings with those of more representative close-to-patient in vitro models that more accurately reflect the response of EAC cells to chemotherapy. Our proteomic analysis was conducted with a single representative NOF/CAF pair, and further validation of these findings might be sensible. Finally, we have not tested PDE5i efficacy in a spontaneous EAC animal model. Unfortunately, no good model of EAC exists, meaning that the best testing-ground for PDE5i in EAC will be in humans. To mitigate this, we have used a validated near-patient PDX model to demonstrate efficacy.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

ACKNOWLEDGMENTS
The authors would like to thank the core facilities that contributed to this work: the Research Histology at Department of Cellular Pathology, Southampton General Hospital for conducting histology and immunohistochemistry experiments; the Biomedical Imaging Unit at Southampton General Hospital for use of their bioimaging equipment and expertise, namely in fluorescent laser-scanning confocal microscopy and digital slide scanning. Gene expression data in Figure 1C ewers partly based on data generated by the TCGA Research Network: https://www.cancer.gov/tcga. The data used for the analyses described in this manuscript were obtained from the Genotype-Tissue Expression (GTEx) Project is CSO of Proteas Bioanalytics, Inc. They confirm that they were not affiliated when the work published in this study was carried out. All other authors declare no competing interests.

INCLUSION AND DIVERSITY
One or more of the authors of this paper self-identifies as an underrepresented ethnic minority in science. One or more of the authors of this paper self-identifies as a member of the LGBTQ+ community. One or more of the authors of this paper self-identifies as living with a disability. Primary fibroblast cultures Tissue was collected and stored with ethical agreement and informed consent (REC: 09/H0504/66 and 18/NE/0234) at University Hospital Southampton, and fibroblasts were extracted from normal esophagus and esophageal adenocarcinoma and sub-cultured as previously described. 64

Mouse models
The in vivo experiments were conducted under the UK Home Office Licence number PPL P435A9CF8. LASA good practice guidelines, FELASA working group on pain and distress guidelines and ARRIVE reporting guidelines were also followed. All mice were purchased from Charles River UK. Mice were maintained in individually Ventilated Cages (Tecniplast UK) within a barriered unit, illuminated by fluorescent lights set to give a 12 hour light-dark cycle (on 07.00, off 19.00), as recommended in the guidelines to the Home Office Animals (Scientific Procedures) Act 1986 (UK). The room was air-conditioned by a system designed to maintain an air temperature range of 21 ± 2 C and a humidity of 55% ± 10%. Mice were housed in social groups, 3 per cage, during the study, with irradiated bedding and autoclaved nesting materials and environmental enrichment (Datesand UK). Sterile irradiated 5V5R rodent diet (IPS Ltd, UK) and irradiated water (Baxter, UK) was offered ad libitum. The condition of the animals was monitored throughout the study by an experienced animal technician. After a week's acclimatisation, the mice were initiated with tumors as described in the dose-finding study, dose escalation study and efficacy study as described in method details. Dose-finding study for ECX administration in non-tumor bearing mice 3 female CD-1 mice received escalating doses of ECX cycle in 3 phases separated by a drug holiday of 14 days to allow recovery between cycles. Doses were calculated by converting from human standard of care regimens as described in Table S4. Dosing was as follows, with the exception of one mouse that underwent a repeat of cycle 2 (50% clinical equivalent dose) due to slower recovery. Dose cycle 3 is equivalent to 75% of the clinical equivalent ECX dose.
Dose escalation study of ECX in patient-derived xenograft-bearing mice 10 male 8-9 week old CD-1 NuNu mice were implanted with 1x10 6 OES127 cells re-suspended in 100 ml of Matrigel (Corning), which were developed from an esophageal adenocarcinoma resection specimen + eGFP labelled mesenchymal stem cells (MSCs) in a ratio of 2:1. The cells were generated by disaggregating from donor OES127 PDXs by a collagenase/dispase disaggregation fluid, and rotating at 37 C for 1 hour, counted and viability measured by trypan blue, before resuspending both the MSCs and PDX simultaneously in matrigel. These were injected subcutaneously into the left flank of the mice and the resulting tumours were measured twice weekly using Vernier calipers and the volumes calculated using the formula V=ab2/6, where a is the length and b is the width. A secondary dose of MSCs, this time lentivirally transduced with pLVX-fLuc, were added as a 'boost', 14 days after initiation, directly injected into the tumor in Phosphate Buffered Saline (Sigma, UK). Dosing commenced on day 18 post-initiation and followed the regime below, with a 14 day rest period between each cycle for observation of side effects, during which time animals were weighed daily.
Dosing *IV=intravenous, IP=intraperitoneal, PO=per os, SFI=saline for injection, WFI= water for injection As this was a dose escalation tolerability study, no power calculation was required, groups 1 and 2 having 2 mice each and groups 3 and 4 having 3 mice each. The mice were terminated between days 42-56 due to tumors approaching maximum allowable size. They were culled by cervical dislocation, tumors were dissected out and weighed, aortas, hearts, livers and spleens were also dissected out, and all were fixed in Neutral Buffered Formalin.
Due to some slight weight loss in one group (ECX) during this dose escalation study which was thought to be associated with the use of epirubicin, an additional tolerability study was carried out using cisplatin and capecitabine only, at 75% dose of those used in the Dose Escalation (Cisplatin 2.25 mg/kg, IP, days 1, 3, 5 + Capecitabine 75 mg/kg, PO, days 1, 2, 3, 4, 5) and using the PDE5is daily (instead of twice daily at lower doses). This dosing regime, which was well-tolerated was adopted for the Efficacy study (see details below).
Efficacy study of PDE5i and CX in patient-derived xenograft-bearing mice 60 female 7-8 week old CD-1 NuNu mice were used for PDX experiments, testing CX treatment in combination with PDE5i, vardenafil or tadalafil. Patient-derived xenografts were developed using OES127 cells and eGFP-labelled hMSCs as above. Due to loss of the human stromal compartment in such models, human mesenchymal stem cells (hMSCs) were co-implanted with the xenograft and supplemented before treatment began as before. Tumors were measured as previously detailed, and mice were weighed weekly. Tumors were also imaged weekly in the IVISâ Spectrum imaging system (PerkinElmer, MA, USA) by 2D optical imaging, with tumor measurements made using Living Image (4.3.1) software and standard open filters to assess the retention of MSCs. Prior to imaging, the mice were anaesthetised with an injectable anaesthetic combination (Anaestemine [ketamine]/Sedastart [medetomadine], Animalcare Ltd. UK) before being placed in the IVIS system and imaged on days 0, 1, 8, 15, 16, 21, 23 and 28, mice being allowed to recover from the anaesthetic with appropriate post procedural monitoring and therapy, including placing mice on a heat pad and providing fluid replacement via wet mash once awake.
On day 14 after tumor initiation, the mice were randomised into one of four groups (n=15/group) by tumor size and fluorescence. Power calculations to determine group sizes were based on One way ANOVA with 4 groups, to allow detection of a 40% effect of treatment at a power of 80%. This gave a minimum required sample size of n=10 per group but based on a potential 70% take rate (due to the tissue being PDX in origin and the implant location) sample size was increased to n=15 per group. Dosing followed the weekly cycle below for 3 weeks, with 2 days dosing in week 4 prior to termination one week after final dosing.
Dosing regime: The mice were culled by cervical dislocation, tumors were dissected out and weighed, before half was snap frozen in liquid nitrogen, half was fixed in Neutral Buffered Formalin and processed for paraffin embedding. Data from one of the mice in Group 2 was excluded from the final analysis because it reached the maximum allowed size a week before any of the other mice in the study needed to be terminated, and thus was considered an outlier. Growth of tumors was assessed based on caliper measurements and expressed as a percentage of the pre-treatment volume for individual mice. Mean and standard error was calculated for each group and analysed by 2-way ANOVA to compare each group to the untreated group and by Kruskal-Wallis test to compare relative tumor volume between the groups at the final timepoint.